Specific binding agents

ABSTRACT

A reshaped human antibody or reshaped human antibody fragment having specificity for human polymorphic epithelial mucin (PEM) is produced by transferring the complementarity determining regions (CDRs) from a murine anti-HMFG hybridoma cell line HMFG1 into a human antibody variable region framework. The reshaped molecule can be used in the treatment or diagnosis of cancer.

[0001] This invention relates to specific binding agents, and inparticular to polypeptides containing amino acid sequences that bindspecifically to other proteinaceous or non-proteinaceous materials. Theinvention most particularly concerns the production of such specificbinding agents by genetic engineering.

[0002] Antibody Structure

[0003] Natural antibody molecules consist of two identical heavy-chainand two identical light-chain polypeptides, which are covalently linkedby disulphide bonds. FIG. 14 of the accompanying drawingsdiagramatically represents the typical structure of an antibody of theIgG class. Each of the chains is folded into several discrete domains.The N-terminal domains of all the chains are variable in sequence andtherefore called the variable regions (V-regions). The V-regions of oneheavy (VH) and one light chain (VL) associate to form theantigen-binding site. The module formed by the combined VH and VLdomains is referred to as the Fv (variable fragment) of the antibody.The C-terminal ends of both heavy and light chains are more conserved insequence and therefore referred to as the constant regions. Heavy chainconstant regions are composed of several domains, eg. the heavy chainconstant region of the gamma-isotype (IgG) consists of three domains(CH1, CH2, CH3) and a hinge region which connects the CH1 and CH2domains. The hinges of the two heavy chains are covalently linkedtogether by disulphide bridges. Light chains have one constant domainwhich packs against the CH1 domain. The constant regions of the antibodymolecule are involved in effector functions such as complement lysis andclearing by Antibody Dependant Cell Cytotoxicity (ADCC). Classicaldigestion of an antibody with the protease papain yields threefragments. One fragment contains the CH2 and CH3 domains and, as itcrystallises easily, was called the Fc fragment. The other two fragmentswere designated the Fab (antigen-binding) fragments, they are identicaland contain the entire light chain combined with the VH and CH1 domain.When using pepsin, the proteolytic cleavage is such that the two Fabsremain connected via the hinge and form the (Fab)₂ fragment. Each of thedomains is represented by a separate exon at the genetic level.

[0004] The variable regions themselves each contain 3 clusters ofhypervariable residues, in a framework of more conserved sequences.These hypervariable regions interact with the antigen, and are calledthe Complementarity Determining Regions (CDRs). The more conservedsequences are called the Framework Regions (FRs). See Kabat et al(1987). X-ray studies of antibodies have shown that the CDRs form loopswhich protrude from the top of the molecule, whilst the FRs provide astructural beta-sheet framework.

[0005] Modified Antibodies

[0006] In one embodiment, the invention relates to so-called “reshaped”or “altered” human antibodies, ie. immunoglobulins having essentiallyhuman constant and framework regions but in which the complementaritydetermining regions (CDRs) correspond to those found in a non-humanimmunoglobulin, and also to corresponding reshaped antibody fragments.

[0007] The general principles by which such reshaped human antibodiesand fragments may be produced are now well-known, and reference can bemade to Jones et al (1986), Riechmann et al (1988), Verhoeyen et al(1988), and EP-A-239400 (Winter). A comprehensive list of relevantliterature references is provided later in this specification.

[0008] Reshaped human antibodies and fragments have particular utilityin the in-vivo diagnosis and treatment of human ailments because theessentially human proteins are less likely to induce undesirable adversereactions when they are administered to a human patient, and the desiredspecificity conferred by the CDRs can be raised in a host animal, suchas a mouse, from which antibodies of selected specificity can beobtained more readily. The variable region genes can be cloned from thenon-human antibody, and the CDRs grafted into a human variable-regionframework by genetic engineering techniques to provide the reshapedhuman antibody or fragment. To achieve this desirable result, it isnecessary to identify and sequence at least the CDRs in the selectednon-human antibody, and preferably the whole non-human variable regionsequence, to allow identification of potentially important CDR-frameworkinteractions.

[0009] Antibodies raised against the human milk fat globule (HMFG),generally in a delipidated state, can exhibit a broad spectrum ofreactivity with epithelial origin neoplasms, particularly carcinomas ofthe breast, ovary, uterus and lung. See Taylor-Papadimitriou et al(1981) and Arklie et al (1981). One well-characterised antibody(designated HMFG1) is known to bind to a component of the HMFG, alsofound in some body tissues, some cancer tissues and urine, which hasbeen designated polymorphic epithelial mucin (PEM) (Gendler et al,1988). Binding is thought to involve the peptide core of the PEM.Corresponding useful specificity can be achieved by raising antibodiesagainst cancer cells, for example breast cancer cell lines.

[0010] EP-A2-0369816 (The University of Melbourne, Xing et al) describesmonoclonal antibodies specific for human polymorphic epithelial mucin,which bind to a defined amino acid sequence. It is suggested inEP-A2-0369816 that the described antibodies may be “humanised” accordingto the method of Riechmann et al (1988). However, Xing et al do notdescribe the actual preparation of any such reshaped anti-PEMantibodies.

SUMMARY OF THE INVENTION

[0011] The invention provides, as one embodiment, a synthetic specificbinding polypeptide having specificity for a polymorphic epithelialmucin (PEM), and especially a synthetic specific binding polypeptidehaving anti-human milk fat globule (HMFG) specificity, containing one ormore of the CDRs depicted in FIGS. 1 and 2 of the accompanying drawings.By synthetic, we particularly mean that the polypeptide is produced byrecombinant DNA technology, and to that extent at least is differentfrom a naturally-occurring or naturally-induced specific binding agenthaving identical specificity. Alternatively, the synthetic polypeptidehas been produced by artificially assembling a sequence of amino acidsto produce a novel or nature-identical molecule. The syntheticpolypeptide can be equivalent to an intact conventional antibody, orequivalent to a multiple or single-chain fragment of such an antibody,or can be simply a material that includes one or more sequences thatconfer the desired specific binding capability.

[0012] The invention provides as an important embodiment a reshapedhuman antibody, or a reshaped human antibody fragment, having anti-PEMspecificity, and especially having anti-HMFG specificity, containing oneor more of the CDRs depicted in FIGS. 1 and 2 of the accompanyingdrawings. Preferably, the reshaped antibody or fragment of the inventioncontains all 3 of the CDRs depicted in FIG. 1 of the accompanyingdrawings, in a human heavy chain variable region framework.Alternatively, or in addition, the reshaped antibody or fragment of theinvention contains all 3 of the CDRs depicted in FIG. 2 of theaccompanying drawings, in a human light chain variable region framework.

[0013] Another embodiment of the invention is a reshaped antibody orreshaped antibody fragment containing a protein sequence as depicted inFIG. 12 and/or FIG. 13 of the accompanying drawings.

[0014] Other important embodiments of the invention are an expressionvector incorporating a DNA sequence as depicted in FIG. 12 and/or FIG.13 of the accompanying drawings, and an expression vector incorporatinga DNA sequence encoding one or more of the protein sequences designatedas being a CDR in FIG. 1 and/or FIG. 2 of the accompanying drawings.

[0015] An important aspect of the invention is a stable host cell linecontaining a foreign gene that causes the host cell line to produce aspecific binding agent according to the invention. This can be a stablehost cell line containing a foreign gene that encodes at least one ofthe amino acid sequences designated as being a CDR in FIG. 1 and/or FIG.2 of the accompanying drawings, together with a protein framework thatenables the encoded amino acid sequence when expressed to function as aCDR having specificity for HMFG.

[0016] The invention also provides an immortalised mammalian cell line,or a yeast, or other eukaryotic cell, or a prokaryotic cell such as abacterium, producing a reshaped antibody or fragment according to theinvention.

[0017] Another important aspect of the invention is a synthetic specificbinding agent, reshaped human antibody or reshaped human antibodyfragment, having specificity equivalent to that of the gamma-1, kappaanti-HMFG monoclonal antibody “HMFG1”.

[0018] The invention also provides two novel plasmids,pSVgpt-HuVHHMFG1-HuIgG1 and pSVneo-HuVkHMFG1-HuCk, and these plasmidscan be used in the production of a synthetic specific binding agent,reshaped human antibody or reshaped human antibody fragment.

[0019] These plasmids are contained in novel E.coli strains NCTC 12411and NCTC 12412, respectively.

[0020] Other aspects of the invention are:

[0021] a) A DNA sequence encoding a reshaped human antibody heavy-chainvariable region having specificity for HMFG, as contained in E.coli NCTC12411.

[0022] b) A DNA sequence encoding a reshaped human antibody light-chainvariable region having specificity for HMFG, as contained in E.coli NCTC12412.

[0023] c) A reshaped human antibody heavy-chain variable region havingspecificity for HMFG, producible by means of the expression vectorcontained in E.coli NCTC 12411.

[0024] d) A reshaped human antibody light-chain variable region havingspecificity for HMFG, producible by means of the expression vectorcontained in E.coli NCTC 12412.

[0025] e) A reshaped human antibody or reshaped human antibody fragment,comprising at least one variable region according to c) or d) above.

[0026] A particular embodiment of the invention is therefore a reshapedhuman antibody or reshaped human antibody fragment possessing anti-HMFGspecificity and incorporating a combination of CDRs (which may, forexample, be cloned from a murine anti-HMFG immunoglobulin) having theamino acid sequences identified as CDR1, CDP2 and CDR3 respectively inFIGS. 1 and 2 of the accompanying drawings, which respectively representthe heavy chain variable region (VH) and light chain variable region(Vk) of a murine anti-HMFG monoclonal antibody that we have cloned andsequenced. In the case of an intact antibody, or a fragment comprisingat least one heavy chain variable region and at least one light chainvariable region, the reshaped antibody or fragment preferably containsall six CDRs from the non-human source. To be most effective in binding,the CDRs should preferably be sited relative to one another in the samearrangement as occurs in the original non-human antibody, e.g. the VHCDRs should be in a human VH framework, and in the order in which theyoccur naturally in the non-human antibody.

[0027] As will be apparent to those skilled in the art, the CDRsequences and the surrounding framework sequences can be subject tomodifications and variations without the essential specific bindingcapability being significantly reduced. Such modifications andvariations can be present either at the genetic level or in the aminoacid sequence, or both. Accordingly, the invention encompasses synthetic(reshaped) antibodies and fragments that are functionally equivalent tothose described herein having precisely defined genetic or amino acidsequences.

[0028] The invention can also be applied in the production ofbi-specific antibodies, having two Fab portions of differentspecificity, wherein one of the specificities is conferred by a reshapedhuman variable chain region incorporating one or more of the CDRsdepicted in FIGS. 1 and 2 of the accompanying drawings.

[0029] The invention can also be applied in the production of so-calledsingle-chain antibodies (for example, as disclosed in GenexEP-A-281604), and also to polysaccharide-linked antibodies (seeHybritech EP-A-315456), and other modified antibodies.

[0030] Any human constant regions (for example, gamma 1, 2, 3 or 4-type)can be used.

[0031] Antibody fragments retaining useful specific binding propertiescan be (Fab)₂, Fab, Fv, VH or Vk fragments. These can be derived from anintact reshaped antibody, for example by protease digestion, or producedas such by genetic engineering.

[0032] Practical Applications of the Invention

[0033] An important aspect of the invention is a reshaped humananti-HMFG antibody or fragment, as defined above, linked to orincorporating an agent capable of retarding or terminating the growth ofcancerous cells, or to an imaging agent capable of being detected whileinside the human body. The invention also includes injectablecompositions comprising either of such combinations in apharmaceutically acceptable carrier, such as saline solution, plasmaextender or liposomes. The invention also includes the use, in a methodof human cancer therapy or imaging, of a reshaped human anti-HMFGantibody or fragment as defined above. The invention further includesthe use of such an antibody or fragment for the manufacture of amedicament for therapeutic application in the relief of cancer inhumans, or the use of such an antibody or fragment in the manufacture ofa diagnostic composition for in-vivo diagnostic application in humans.

[0034] The Fc region of the antibody, itself using pathways andmechanisms available in the body, such as complement lysis and antibodydependent cellular cytotoxicity, can be used to affect adversely thegrowth of cancerous cells. In this embodiment, no additional reagentneed be linked to the reshaped antibody.

[0035] Examples of agents capable of affecting adversely the growth ofcancerous cells include radioisotopes, such as Yttrium 90 and Iodine131; drugs such as methotrexate; toxins such as ricin or parts thereof;and enzymes which may for example turn an inactive drug into an activedrug at the site of antibody binding.

[0036] Examples of imaging agents include radioisotopes generating gammarays, such as Indium 111 and Technetium 99; radioisotopes generatingpositrons, such as Copper 64; and passive agents such as Barium whichact as contrast agents for X-rays, and Gadolinium in nmr/esr scanning.

[0037] In order to link a metallic agent, such as a radioisotope, to aspecific binding agent of the invention, it may be necessary to employ acoupling or chelating agent. Many suitable chelating agents have beendeveloped, and reference can be made for example to U.S. Pat. No.4,824,986, No. 4,831,175, No. 4,923,985 and No. 4,622,420. Techniquesinvolving the use of chelating agents are described, for example, inU.S. Pat. No. 4,454,106, No. 4,722,892, Moi et al (1988), McCall et al(1990), Deshpande et al (1990) and Meares et al (1990).

[0038] The use of radiolabelled antibodies and fragments in cancerimaging and therapy in humans is described for example in EP 35265. Itmay be advantageous to use the radiolabelled cancer-specific antibody orfragment in conjunction with a non-specific agent radiolabelled with adifferent isotope, to provide a contrasting background for so-calledsubtraction imaging.

[0039] The antibody reagents of the invention can be used to identify,e.g. by serum testing or imaging, and/or to treat, PEM-producingcancers. Such cancers can occur as for example, carcinomas of breast,ovary, uterus and lung, or can manifest themselves as liquids such aspleural effusions.

[0040] Modified Antibody Production

[0041] The portions of the VH and VL regions that by convention (Kabat,1987) are designated as being the CDRs may not be the sole features thatneed to be transferred from the non-human monoclonal antibody.Sometimes, enhanced antibody performance, in terms of specificity and/oraffinity, can be obtained in the reshaped human antibody if certainnon-human framework sequences are conserved in the reshaped humanantibody. The objective is to conserve the important three-dimensionalprotein structure associated with the CDRs, which is supported bycontacts with framework residues.

[0042] The normal starting point from which a reshaped antibody inaccordance with the invention can be prepared, is a cell (preferably animmortalised cell line), derived from a non-human host animal (forexample, a mouse), which expresses an antibody having specificityagainst HMFG or PEM. Such a cell line can, for example, be a hybridomacell line prepared by conventional monoclonal antibody technology.Preferably, the expressed antibody has a high affinity and highspecificity for HMFG, because it should be anticipated that some loss ofaffinity and/or specificity may occur during the transfer of theseproperties to a human antibody or fragment by the procedures of theinvention. By selecting a high specificity antibody as the parentantibody, the likelihood that the final reshaped antibody or fragmentwill also exhibit effective binding properties is enhanced.

[0043] The next stage is the cloning of the cDNA from the cellexpressing the selected non-human antibody, and sequencing andidentification of the variable region genes including the sequencesencoding the CDRs. The experimental procedures involved can now beregarded as routine in the art, although they are still laborious.

[0044] If the object is to produce a reshaped complete human antibody,or at least a fragment of such an antibody which will contain both heavyand light variable domains, it will be necessary to sequence the cDNAassociated with both of these domains.

[0045] Once the relevant cDNA sequence or sequences have been analysed,it is necessary to prepare one or more replicable expression vectorscontaining a DNA sequence which encodes at least a variable domain of anantibody, which variable domain comprises human framework regionstogether with one or more CDRs derived from the selected non-humananti-HMFG antibody. The DNA sequence in each vector should includeappropriate regulatory sequences necessary to ensure efficienttranscription and translation of the gene, particularly a promoter andleader sequence operably linked to the variable domain sequence. In atypical procedure to produce a reshaped antibody or fragment inaccordance with the invention, it may be necessary to produce two suchexpression vectors, one containing a DNA sequence for a reshaped humanlight chain and the other, a DNA sequence for a reshaped human heavychain. The expression vectors should be capable of transforming a chosencell line in which the production of the reshaped antibody or fragmentwill occur. Such a cell line may be for example, a stable non-producingmyeloma cell line, examples (such as NS0 and sp2-0) of which are readilyavailable commercially. An alternative is to use a bacterial system,such as E.coli, as the expression vehicle for the reshaped antibody orfragment. The final stages of the procedure therefore involvetransforming the chosen cell line or organism using the expressionvector or vectors, and thereafter culturing the transformed cell line ororganism to yield the reshaped human antibody or fragment.

[0046] By way of example only, detailed steps by means of whichappropriate expression vectors can be prepared are given later in thisspecification. The manipulation of DNA material in a suitably equippedlaboratory is now a well-developed art, and the procedures required arewell within the skill of those versed in this art. Many appropriategenomic and cDNA libraries, plasmids, restriction enzymes, and thevarious reagents and media which are required in order to perform suchmanipulations, are available commercially from suppliers of laboratorymaterials. For example, genomic and cDNA libraries can be purchased fromClontech Laboratories Inc. The steps given by way of example below arepurely for the guidance of the reader of this specification, and theinvention is in no way critically dependant upon the availability of oneor more special starting materials. In practice, the skilled person hasa wide range of materials from which to choose, and can exploit andadapt the published technology using acquired experience and materialsthat are most readily available in the scientific environment. Forexample, many plasmids fall into this category, having been so widelyused and circulated within the relevant scientific community that theycan now be regarded as common-place materials.

EXAMPLES

[0047] The procedure used to prepare reshaped anti-HMFG human antibodiesis described in detail below, by way of example only, with reference tothe accompanying drawings, of which:

[0048]FIG. 1 shows the cDNA sequence coding for a murine heavy chainvariable region having anti-HMFG specificity. The 3 classical CDRs areindicated, together with an amino acid sequence matching the cDNA code.

[0049]FIG. 2 shows the cDNA sequence coding for a murine light chainvariable region having anti-HMFG specificity.

[0050]FIG. 3a shows a design for a synthesic reshaped human VH gene withHMFG1 specificity (HuVHIconHMFG1 gene cassette) containing 3 fragments.

[0051]FIGS. 3b to 3 d show the sequence of the respective fragments inFIG. 3a, and also the oligonucleotides used in the assembly of eachfragment.

[0052]FIGS. 4a, 4 b and 4 c together show a route by which an expressionvector encoding a reshaped human heavy chain incorporating the CDRs ofFIG. 1, can be prepared.

[0053]FIGS. 5a and 5 b together show a similar transformation route toobtain an expression vector encoding a reshaped human light chainincorporating the CDRs of FIG. 2, can be prepared.

[0054]FIG. 6 shows the plasmid pUC12-IgEnh, which contains an enhancersequence used in the routes of FIGS. 4a to 5 b.

[0055]FIG. 7 shows the source of plasmid pBGS18-HulgG1 used in the routeof FIG. 4c.

[0056]FIG. 8 shows the source of plasmid pBGS18-HuCk used in the routeof FIG. 5b.

[0057]FIG. 9 shows two synthetic oligonucleotide sequences I and II usedin cloning the cDNA sequences of FIGS. 1 and 2.

[0058]FIG. 10 shows two synthetic oligonucleotide sequences III and IVused to introduce the Kpn I and Sal I restriction sites in M13mp9HuVHLYSrespectively, in the route depicted in FIG. 4a.

[0059]FIG. 11 shows three synthetic oligonucleotide sequences VI, VIIand VIII used to graft the Vk HMFG1 CDRs onto the human VK REI frameworkregions in the route depicted in FIG. 5a.

[0060]FIGS. 12 and 13 show the cDNA and amino acid sequences of theresulting reshaped human heavy and light chain variable regionsrespectively.

[0061]FIG. 14 depicts in diagramatic form the structure of a typicalantibody (immunoglobulin) molecule.

[0062]FIG. 15 shows in graphical form the relative specific anti-HMFG1binding activity of the resulting reshaped human antibody.

[0063] The experimental procedures required to practice the invention donot in themselves represent unusual technology. The cloning andmutagenesis techniques were performed as generally described for examplein Verhoeyen et al (1988); Riechmann et al (1988) and EP-A-239400(Winter). The “de novo” synthesis of a reshaped human heavy chainvariable region gene (see FIGS. 3a-3 d) was done by conventionaltechniques, using a set of long overlapping oligonucleotides (see alsoJones et al, 1988). Laboratory equipment and reagents for synthesisinglong oligonucleotides are readily available, and as techniques in thisfield develop it is becoming practicable to synthesise progressivelylonger sequences.

[0064] Detailed laboratory manuals, covering all basic aspects ofrecombinant DNA techniques, are available, e.g. “Molecular Cloning” bySambrook et al (1989).

[0065] By means of the invention, the antigen binding regions of a mouseanti-HMFG antibody (HMFG1) were grafted onto human framework regions.The resulting reshaped human antibody (designated HuHMFG1) has bindingcharacteristics similar to those of the original mouse antibody.

[0066] Such reshaped antibodies can be used for in vivo diagnosis andtreatment of human cancers, eg. ovarian cancers and breast cancers, andare expected at least to reduce the problem of an immune response in thepatient often seen upon administration of non-human antibody. A similarbenefit has been shown for reshaped CAMPATH-1 antibody in Hale et al(1988).

[0067] Methods:

[0068] 1. Cloning and Sequence Determination of the Mouse VariableRegion Genes

[0069] Messenger RNA was isolated from a murine hybridoma line whichsecretes the gamma-1, kappa anti-HMFG antibody “HMFG1” (seeTaylor-Papadimitriou et al, 1981 and Arklie et al, 1981). First strandcDNA was synthesised by priming with oligonucleotides I and II (see FIG.9) complementary to the 5′ ends of the CH1 and Ck exons respectively.Second strand cDNA was obtained as described by Gübler and Hoffmann(1983).

[0070] Kinased EcoRI linkers were ligated to the heavy chaindouble-stranded cDNA and Pst1 linkers to the light chain double-strandedcDNA (both were first treated with EcoRI or PstI methylase to protectpossible internal sites), followed by cloning into EcoRI or PstI-cutpUC9 (Vieira et al, 1982) and transformation of E.coli strain TG2(Gibson, 1984).

[0071] Colonies containing genes coding for murine HMFG1 VH (MoVHHMFG1)and for murine anti-HMFG Vk (MoVkHMFG1) were identified by colonyhybridisation with 2 probes consisting respectively of 32P-labelledfirst strand cDNA of HMFG1 VH and Vk. Positive clones were characterisedby plasmid preparation, followed by EcoRI or PstI digestion and 1.5%agarose gel analysis. Full-size inserts (about 450 bp) were subcloned inthe EcoRI or PstI site of M13mp18 (Norrander et al, 1983). This yieldedclones with inserts in both orientations, facilitating nucleotidesequence determination of the entire insert, by the dideoxy chaintermination method (Sanger et al, 1977).

[0072] The nucleotide sequences, and their translation into amino acidsequences, of the mature variable region genes MoVHHMFG1 and MoVkHMFG1,are shown in FIGS. 1 and 2. The 450 bp inserts included a signalsequence and 51 untranslated sequences and linkers, not shown in theFigures.

[0073] 2. Grafting of the Mouse HMFG1 CDRs Onto Human Framework Regions

[0074] The general techniques necessary to achieve this have beendescribed very adequately in Jones et al (1986), Verhoeyen et al (1988),Riechmann et al (1988) and in EP-A-239400 (Winter).

[0075] a) Light Chain:

[0076] The basic construct used for reshaping a human light chain wasM13mp9HuVkLYS (Riechmann et al, 1988), which contains framework regionswith sequences based on those of the light chain variable regions of thehuman Bence-Jones protein REI (Epp et al, 1974).

[0077] The CDRs in this construct (FIG. 5a) were replaced bysite-directed mutagenesis with oligonucleotides VI, VII and VIIIencoding the HMFG1 kappa chain CDRs flanked by 12 nucleotides at eachend encoding the corresponding human framework residues. Theseoligonucleotides are shown in FIG. 11. The mutagenesis was done asdescribed in Riechmann et al (1988). The resulting reshaped human lightchain variable region gene (HuVkHMFG1) is shown in FIG. 13.

[0078] b) Heavy Chain:

[0079] A reshaped human heavy chain variable region gene was obtained by“de novo” synthesis. In the experiments published by Jones et al, etc,mentioned above, rodent heavy chain CDRs were grafted onto the frameworkregions of the human NEW heavy chain variable region. It was shown byVerhoeyen et al (1988) and by Riechmann et al (1988) that it isimportant that the human framework can support the rodent CDRs in aconformation similar to the one occurring in the original rodentantibody, and that certain CDR-framework interactions can be critical.It follows thus that the more dissimilar the rodent and the humanframework sequences are, the less the chance will be for the CDR graftto “take”.

[0080] Comparison of the heavy chain variable region amino acid sequenceof the mouse HMFG1 (FIG. 1) to that of the human NEW (as used inVerhoeyen et al, 1988), revealed 44% differences between theirrespective framework regions. A much better homology was found whencomparing to human heavy chain variable regions of subgroup I (Kabat etal, 1987); human VHNEW belongs to subgroup II.

[0081] We therefore decided to synthesise a human heavy chain variableregion gene of subgroup I, containing the HMFG1 heavy chain CDRs. Wedesigned a consensus sequence for human heavy chain subgroup I variableregions, based on sequence information on this subgroup in Kabat et al,1987. Optimal codon usage was taken from the sequences of mouse constantregion genes (the genes are expressed in a mouse myeloma line).

[0082] There are only 14% differences between the framework sequences ofthe HMFG1 VH and the VH of this human VH subgroup I consensus sequence(HuVHIcon). The resulting reshaped gene was designated the nameHuVHIconHMFG1, and is depicted in FIG. 12. The gene synthesis isdescribed separately in section (c) below. The newly synthesised geneHuVHIconHMFG1 was used to replace HuVHLYS in the construct M13mp9HuVHLYS(Verhoeyen et al, 1988), yielding the vector M13mp9HuVHIconHMFG1 (seeFIG. 4a).

[0083] 3. Assembly of Reshaped Human Antibody Genes in ExpressionVectors

[0084] The next stage involved the use of a murine heavy chain enhancerIgEnh, described in Neuberger et al (1983) where the enhancer iscontained in a 1 kb Xbal fragment of plasmid pSV-Vμl. The 700 bpXbal/EcoRI subfragment of this 1 kb Xbal fragment is sufficient toconfer enhancer activity.

[0085] An alternative source of this enhancer is plasmid pSVneoHuVkPLAP(see FIG. 5a), a variation of which has been deposited in an E.colistrain under the Budapest Treaty on Apr. 19, 1990 as NCTC 12390. Asdeposited, the plasmid also contains a human kappa-chain constant regiongene (cloned in the BamH1 site).

[0086] The reshaped human genes as prepared in sections 2(a) and 2(b)above, were excised from the M13 vectors as HindIII-BamHI fragments. Theheavy chain variable region genes were cloned into a vector based onpSV2gpt (Mulligan et al, 1981) and the light chain variable region genescloned into a vector based on pSV2neo (Southern et al, 1981) expressionvectors, both containing the immunoglobulin heavy chain enhancer IgEnh.In the pSV2gpt based antibody expression vector (see FIGS. 4b-4 c), theXbal/EcoRI enhancer containing fragment was cloned in the unique EcoRIsite of the pSV2gpt vector (after ligating EcoRI linkers to the filledin Xbal end of the fragment).

[0087] In the pSVneo based antibody expression vector (see FIGS. 5a-5b), the 1 kb Xbal enhancer containing fragment was first cloned intopUC12 (Vieira et al, 1982), yielding the plasmid pUC12-IgEnh, see FIG.6. The enhancer can then be cut out as a 700 bp EcoRI/HindIII fragment(either orientation of the enhancer will work). This 700 bpEcoRI/HindIII fragment is present in the plasmid pSVneoHuVkPLAP, that weused to clone the HuVkHMFG1-containing fragment described in section 2a, see FIGS. 5a and 5 b. The HindIII site in the original pSV2neo hadbeen removed. It is possible to use pSV2gpt as an alternative vector forlight chain expression, as in practice there is no need for neoselection.

[0088] The HuVHIconMFG1 gene was linked to a human gamma 1 constantregion (Takahashi et al, 1982), cloned initially as a 8 kb HindIIIfragment into the HindIII site of pBGS18 (Spratt et al, 1986), and thenin the pSV2gpt expression vector as a BamHI fragment (see FIGS. 4c and7). It should be noted that in the Takahashi et al (1982) referencethere is an error in FIG. 1: the last (3′) two sites are BamH1 followedby HindIII, and not the converse. This was confirmed by Flanagan et al(1982).

[0089] The HuVkHMFG1 gene was linked to a human C kappa constant region(Hieter et al, 1980) also cloned in as a BamHI fragment (see FIGS. 5band 8). The source of the human Ck used in FIG. 8 is given in Hieter etal (1980). The 12 kb BamH1 fragment from embryonic DNA (cloned in agamma Ch28 vector system) was subcloned in the BamH1 site of plasmidpBR322.

[0090] 4. “de novo” Synthesis of the HuVHIconHMFG1 Gene

[0091] We decided to synthesise a gene encoding a human variable regiongene of subgroup I (Kabat et al, 1987), and with the CDRs of VHHMFG1(FIG. 1). In summary, the synthetic gene is designed in such a way thatit can substitute the HuVHLYS gene in the existing M13mp9HuVHLYS vector.The M13mp9HuVHLYS was mutagenized to contain a KpnI and SalI site at theappropriate places (see also FIG. 4a), to enable cloning of the newlysynthesized gene as a KpnI-SalI fragment.

[0092] The gene sequence was designed as described above in section 2(b)and is depicted in FIG. 12. To facilitate the substitution of this genefor the HuVHLYS gene in M13mp9HuVHLYS (Verhoeyen et al, 1988, see alsoFIG. 4a), 5′ and 3′ extensions were added to the gene. The 5′ extensioncontains 37 bp of the leader intron and 11 bp of the second half of theleader exon (as in M13mp9HuVHLYS), and has a KpnI site at the very 5′end. The 3′ extension contains 38 untranslated nucleotides (as inM13mp9HuVHLYS) and ends in a SalI site.

[0093] M13mp9HuVHLYS was modified by site directed mutagenesis witholigonucleotides III and IV to contain a KpnI and SalI site at theappropriate places (see FIG. 4a and FIG. 10). This vector was namedM13mp9HuVHLYS(K,S). This enabled cloning of the HuVHIconHMFG1 gene as aKpnI-SalI fragment in KpnI-SalI cut M13mp9HuVHLYS(K,S) vector.

[0094] For practical reasons it was decided to synthesise the gene asthree fragments (cassettes), which were then assembled in one completegene.

[0095] Each fragment contains one of the three VHHMFG1 CDRs, and caneasily be cloned or removed by using the (existing or newly introduced)unique restriction sites (see FIG. 3a). Each fragment was elongated atthe 5′ and 3′ end to create a HindIII and BamHI site respectively, toenable cloning in pEMBL9 (Dente et al, 1983). The coding strand of eachfragment was divided in oligonucleotides with an average length of 33bases. The same was done for the non-coding strand, in such a way thatthe oligonucleotides overlapped approximately 50% with those of thecoding strand.

[0096] The sequences of each fragment and of the oligonucleotides usedfor assembly, are shown in FIGS. 3b, 3 c and 3 d.

[0097] Before assembling the fragments, the 5′ ends of the syntheticoligonucleotides had to be phosphorylated in order to facilitateligation. Phosphorylation was performed as follows: equimolar amounts(50 pmol) of the oligonucleotides were pooled and kinased in 40 μlreaction buffer with 8 units polynucleotide kinase for 30-45 minutes at37° C. The reaction was stopped by heating for 5 minutes at 70° C. andethanol precipitation. Annealing was done by dissolving the pellet in 30μl of a buffer containing: 7 mM TrisCl pH 7.5, 10 mM 2-mercapto-ethanol,5 mM ATP were added. Subsequently the mixture was placed in a waterbathat 65° C. for 5 minutes, followed by cooling to 30° C. over a period of1 hour. MgCl2 was added to a final concentration of 10 mM. T4 DNA-ligase(2.5 units) was added and the mixture was placed at 37° C. for 30 min.(or overnight at 16° C.). After this the reaction mixture was heated for10 minutes at 70° C. After ethanol precipitation the pellet wasdissolved in digestion buffer and cut with HindIII and BamHI. Themixture was separated on a 2% agarose gel and the fragment with a lengthcorresponding to the correctly assembled cassette was isolated byelectro-elution.

[0098] The fragments (1, 2, 3) were ligated in pEMBL9 (cut withHindIII/BamHI), yielding the vectors pUR4107, pUR4108 and pUR4109respectively. The sequence of the inserts was checked by sequenceanalysis (in both orientations). Fragment 1 was isolated from pUR4107 byKpnI/XhoI digestion, whilst fragment 2 was isolated from pUR4108 byXhoI/SacI digestion, after which they were ligated in KpnI/SacI cutpUR4109 in a three-fragment ligation. The resulting plasmid was namedpUR4110 (see FIG. 4a). Sequencing analysis showed that the insertcontained the desired HuVHIconHMFG1 gene. This gene was cloned in apSV2gpt-derived expression vector as depicted in FIGS. 4b and 4 c. Thevector pSVgptMoVHLYS-MoIgG1 (Verhoeyen et al, 1988) was used as thesource of a pSVgpt-based vector containing the IgEnh enhancer.

[0099] 5. Expression in Myeloma Cells

[0100] Co-transfection of the expression plasmidspSVgptHuVHIconHMFG1-HuIgG1 and pSVneoHuVkHMFG1-HuCk (FIGS. 4c and 5 b)into NSO myeloma cells was done by electroporation (Potter et al, 1984),after linearisation with PvuI. Transfectomas were selected inmycophenolic acid containing medium to select for cells expressing thegpt gene product, and screened for antibody production and anti-HMFGactivity by ELISA assays.

[0101] Clones positive for both assays were obtained and subcloned bylimiting dilution and pure clones were assayed again for anti-HMFGactivity, and the best producing clones were grown in serum-free mediumfor antibody production.

[0102] 6. Deposited Plasmids

[0103]E.coli strains containing plasmids used in the above procedurehave been deposited, in accordance with the provisions of the BudapestTreaty, in the National Collection of Type Cultures on Jul. 11, 1990 asfollows:

[0104] NCTC 12411: K12, TG1 E.coli containing plasmidpSVgptHuVHIconHMFG1-HuIgG1 (identified for the purposes of depositionsimply as pSVgpt-HuVHHMFG1-HuIgG1)

[0105] NCTC 12412: K12, TG1 E.coli containing plasmidpSVneo-HuVkHMFG1-HuCk

[0106] 7. Binding Ability of the Reshaped Human Antibodies

[0107] A useful way of demonstrating binding ability of the reshapedantibody is to show that it has a similar antibody dilution curve whenbinding to antigen adsorbed on a solid surface. Such curves weregenerated as follows, using the parent murine anti-HMFG antibody and areshaped human antibody prepared by the foregoing procedure.

[0108] 0.5 ml of 10% w/v M280 tosyl activated magnetic beads (Dynal,Wirral, UK) were coupled to milk mucin (10 units as determined in animmunoassay for HMFG1 in which normal human serum registers 100-200units per ml). Milk mucin was prepared from human breast milk accordingto the method of Burchell et al (1987). The level of mucin was chosen toprovide suitable activity for the assays in which the beads were used.The coupling was in 2.5 ml of 0.5M borate buffer at pH 9.5 plus 2.5 mlof mucin in phosphate-buffered saline pH 7.2 (PBS) for 22 hrs at 37° C.with gentle rotation. Blocking of remaining active sites wasaccomplished by adding 1 ml of 10% bovine serum albumen (BSA; Sigma) inPBSA (PBS+0.02% sodium azide followed by a further 7 hr incubation at37° C. The excess protein was washed away after using a samarium cobaltmagnet to pellet the beads. Further washing was 3× in wash buffer (0.1Mpotassium phosphate pH 8.0, 0.1% Tween 20, 0.5% BSA) and 4× in rinsebuffer (PBS+0.1% BSA, 0.1% merthiolate). Beads were stored in rinsebuffer at 10% w/v (estimated by dry weight analysis).

[0109] Antibody binding was measured from a series of doubling dilutionsof antibody samples (prepared by weighing in critical cases). 50 μlsamples were incubated in replicate in microtitre wells with 50 μl of0.05% w/v suspension of beads in 1% BSA/PBSM (PBS+0.01% merthiolate) atroom temperature for 1 hr on a plate shaker. Small cobalt samariummagnets, embedded in a plastic base, were used to sediment the beads tothe sides of the wells of the plate to allow liquid removal and washingonce with 150 μl PBSTM (PBSM+0.15% Tween 20). This was followed bydetection of bound antibody with 50 μl of alkaline phosphatase coupledgoat anti-human IgG (H+L) (Jackson) used at {fraction (1/1000)} dilutionin 1% BSA in PBSTM for 1 hr at room temperature. The beads were washed3× in PBSTM. Colour development was with 200 μl of nitro phenylphosphate (Sigma alkaline phosphatase substrate tablets) in 1Mdiethanolamine buffer at pH 9.8. Optical densities were read in aDynatech plate reader at 410 nm after transferring fixed volumes ofsupernatant (usually 150 μl) to a flat bottom well microtitre plate. Forexamination of mouse antibodies the conjugate used was rabbit anti-mouseIgG (Sigma).

[0110] Antibody dilution curves for the murine and reshaped HMFG1antibodies are shown in FIG. 15. Maximum binding was determined with alarge excess of antibody and negative controls had none. Antibodyconcentrations, in μg/ml, were determined by UV absorption measurementsat 280 nm. For both antibodies a dilution of 1 has been set equivalentto 1 μg/ml. The two curves are similar, indicating a significant anduseful level of binding effectiveness for the reshaped antibody of theinvention.

REFERENCES

[0111] Arklie et al (1981)—Int. J. Cancer, 28, p.23-29

[0112] Burchell et al (1987)—Cancer Res., 47, p.5476

[0113] Dente et al (1983)—Nucleic Acids Res. II, p.1645-1655

[0114] Epp et al (1974)—Eur. J. Biochem. 45, p.513-524

[0115] Flanagan et al (1982)—Nature, 300, p.709-713

[0116] Gendler et al (1988)—J. Biol. Chem, 236, p.12820-12823

[0117] Gibson T (1984)—PhD thesis, LMB-MRC Cambridge

[0118] Gubler et al (1983)—Gene, 25, p.263-269

[0119] Hale et al (1988)—Lancet, 2, p.1394

[0120] Hieter et al (1980)—Cell, 22, p.197-207

[0121] Jones et al (1986)—Nature, 321, p.522-525

[0122] Kabat et al (1987)—in Sequences of Proteins of ImmunologicalInterest, p.ix -US Dept of Health and Human Services

[0123] Mulligan et al (1981)—Proc. natn. Acad. Sci. U.S.A., 78p.2072-2076

[0124] Neuberger et al (1983)—EMBO Journal, 2, p.1373-1378

[0125] Norrander et al (1983)—Gene, 26, p.101-106

[0126] Potter et al (1984)—PNAS, 81, p.7161-7163

[0127] Riechmann et al (1988)—Nature, 332, p.323-327

[0128] Sambrook et al (1989)—Molecular Cloning, 2nd Edition, Cold SpringHarbour Laboratory Press, New York

[0129] Sanger et al (1977)—PNAS USA, 74, p.5463-5467

[0130] Saul et al (1978)—J. biol. Chem. 253, p.585-597

[0131] Southern et al (1981)—J. molec. appl. Genetics, 1 p.327-345

[0132] Spratt et al (1936)—Gene, 41, p.337-342

[0133] Takahashi et al (1982)—Cell, 29, p.671-679

[0134] Taylor-Papadimitrion et al (1981)—Int. J. Cancer, 28, p.17-21

[0135] Verhoeyen et al (1988)—Science, 239, p.1534-1536

[0136] Vieira et al (1982)—Gene, 19, p.259-268

[0137] Winter (1987)—EP-A-239400

[0138] Xing et al (1990)—EP-A2-369816

1 62 5 amino acids amino acid single linear peptide 1 Ala Tyr Trp IleGlu 1 5 17 amino acids amino acid single linear peptide 2 Glu Ile LeuPro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Ly 1 5 10 15 Gly 9 aminoacids amino acid single linear peptide 3 Ser Tyr Asp Phe Ala Trp Phe AlaTyr 1 5 17 amino acids amino acid single linear cDNA 4 Lys Ser Ser GlnSer Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Le 1 5 10 15 Ala 7 aminoacids amino acid single linear peptide 5 Trp Ala Ser Thr Arg Glu Ser 1 59 amino acids amino acid single linear peptide 6 Gln Gln Tyr Tyr Arg TyrPro Arg Thr 1 5 15 base pairs nucleic acid single linear cDNA 7GCCTACTGGA TAGAG 15 51 base pairs nucleic acid single linear cDNA 8GAGATTTTAC CTGGAAGTAA TAATTCTAGA TACAATGAGA AGTTCAAGGG C 51 27 basepairs nucleic acid single linear cDNA 9 TCCTACGACT TTGCCTGGTT TGCTTAC 2751 base pairs nucleic acid single linear cDNA 10 AAGTCCAGTC AGAGCCTTTTATATAGTAGC AATCAAAAGA TCTACTTGGC C 51 21 base pairs nucleic acid singlelinear cDNA 11 TGGGCATCCA CTAGGGAATC T 21 27 base pairs nucleic acidsingle linear cDNA 12 CAGCAATATT ATAGATATCC TCGGACG 27 354 base pairsnucleic acid both linear cDNA 13 CAGGTTCAGC TGCAGCAGTC TGGAGCTGAGCTGATGAAGC CTGGGGCCTC AGTGAAGATA 60 TCCTGCAAGG CTACTGGCTA CACATTCAGTGCCTACTGGA TAGAGTGGGT AAAGCAGAG 120 CCTGGACATG GCCTTGAGTG GATTGGAGAGATTTTACCTG GAAGTAATAA TTCTAGATA 180 AATGAGAAGT TCAAGGGCAA GGCCACATTCACTGCTGATA CATCCTCCAA CACAGCCTA 240 ATGCAACTCA GCAGCCTGAC ATCTGAGGACTCTGCCGTCT ATTACTGTTC AAGGTCCTA 300 GACTTTGCCT GGTTTGCTTA CTGGGGCCAAGGGACTCCGG TCACTGTCTC TGCA 354 118 amino acids amino acid single linearprotein 14 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro GlyAl 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser AlaTy 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu TrpIl 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu LysPh 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr AlaTy 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val TyrTyr Cy 85 90 95 Ser Arg Ser Tyr Asp Phe Ala Trp Phe Ala Tyr Trp Gly GlnGly Th 100 105 110 Pro Val Thr Val Ser Ala 115 342 base pairs nucleicacid both linear protein 15 GACATTGTGA TGTCACAGTC TCCATCCTCC CTAGCTGTGTCAGTTGGAGA GAAGGTTACT 60 ATGAGCTGCA AGTCCAGTCA GAGCCTTTTA TATAGTAGCAATCAAAAGAT CTACTTGGC 120 TGGTACCAGC AGAAACCAGG GCAGTCTCCT AAACTGCTGATTTACTGGGC ATCCACTAG 180 GAATCTGGGG TCCCTGATCG CTTCACAGGC GGTGGATCTGGGACAGATTT CACTCTCAC 240 ATCAGCAGTG TGAAGGCTGA AGACCTGGCA GTTTATTACTGTCAGCAATA TTATAGATA 300 CCTCGGACGT TCGGTGGAGG CACCAAGCTG GAAATCAAAC GG342 114 amino acids amino acid single linear cDNA 16 Asp Ile Val Met SerGln Ser Pro Ser Ser Leu Ala Val Ser Val Gl 1 5 10 15 Glu Lys Val Thr MetSer Cys Lys Ser Ser Gln Ser Leu Leu Tyr Se 20 25 30 Ser Asn Gln Lys IleTyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gl 35 40 45 Ser Pro Lys Leu LeuIle Tyr Trp Ala Ser Thr Arg Glu Ser Gly Va 50 55 60 Pro Asp Arg Phe ThrGly Gly Gly Ser Gly Thr Asp Phe Thr Leu Th 65 70 75 80 Ile Ser Ser ValLys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gl 85 90 95 Tyr Tyr Arg TyrPro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Il 100 105 110 Lys Arg 240base pairs nucleic acid both linear cDNA 17 ACAGTAGCAG GCTTGAGGAAAGCTTCTATA TATGGGTACC AATGACATCC ACTTTGCCTT 60 TCTCTCCACA GGTGTCCACTCCCAGGTGCA GCTGGTGCAG TCTGGGGCAG AGGTGAAAA 120 GCCTGGGGCC TCAGTGAAGGTCTCCTGCAA GGCTTCTGGC TACACCTTCA GTGCCTACT 180 GATAGAGTGG GTGCGCCAGGCTCCAGGAAA GGGCCTCGAG TGGGTCGGAT CCAGGGAGA 240 32 base pairs nucleicacid single linear cDNA 18 AGCTTCTATA TATGGGTACC AATGACATCC AC 32 33base pairs nucleic acid single linear cDNA 19 TTTGCCTTTC TCTCCACAGGTGTCCACTCC CAG 33 36 base pairs nucleic acid single linear cDNA 20GTGCAGCTGG TGCAGTCTGG GGCAGAGGTG AAAAAG 36 33 base pairs nucleic acidsingle linear cDNA 21 CCTGGGGCCT CAGTGAAGGT GTCCTGCAAG GCT 33 36 basepairs nucleic acid single linear cDNA 22 TCTGGCTACA CCTTCAGTGCCTACTGGATA GAGTGG 36 37 base pairs nucleic acid single linear cDNA 23GTGCGCCAGG CTCCAGGAAA GGGCCTCGAG TGGGTCG 37 40 base pairs nucleic acidsingle linear cDNA 24 GAGAAAGGCA AAGTGGATGT CATTGGTACC CATATATAGA 40 36base pairs nucleic acid single linear cDNA 25 CTGCACCAGC TGCACCTGGGAGTGGACACC TGTGGA 36 33 base pairs nucleic acid single linear cDNA 26TGAGGCCCCA GGCTTTTTCA CCTCTGCCCC AGA 33 33 base pairs nucleic acidsingle linear cDNA 27 GGTGTAGCCA GAAGCCTTGC AGGACACCTT CAC 33 36 basepairs nucleic acid single linear cDNA 28 AGCCTGGCGC ACCCACTCTATCCAGTAGGC ACTGAA 36 29 base pairs nucleic acid single linear cDNA 29GATCCGACCC ACTCGAGGCC CTTTCCTGG 29 180 base pairs nucleic acid bothlinear cDNA 30 GACAGCCGTA GAGTGGGTGC AAGCTTCTCC AGGACTCGAG TGGGTCGGAGAGATTTTACC 60 TGGAAGTAAT AATTCTAGAT ACAATGAGAA GTTCAAGGGC CGAGTGACAGTCACTAGAG 120 CACATCCACA AACACAGCCT ACATGGAGCT CAGCAGCCTG AGGATCCAGCAGCCTGAGG 180 25 base pairs nucleic acid single linear cDNA 31AGCTTCTCCA GGACTCGAGT GGGTC 25 27 base pairs nucleic acid single linearcDNA 32 GGAGAGATTT TACCTGGAAG TAATAAT 27 39 base pairs nucleic acidsingle linear cDNA 33 TCTAGATACA ATGAGAAGTT CAAGGGCCGA GTGACAGTC 39 30base pairs nucleic acid single linear cDNA 34 ACTAGAGACA CATCCACAAACACAGCCTAC 30 20 base pairs nucleic acid single linear cDNA 35ATGGAGCTCA GCAGCCTGAG 20 36 base pairs nucleic acid single linear cDNA36 AGGTAAAATC TCTCCGACCC ACTCGAGTCC TGGAGA 36 39 base pairs nucleic acidsingle linear cDNA 37 GCCCTTGAAC TTCTCATTGT ATCTAGAATT ATTACTTCC 39 24base pairs nucleic acid single linear cDNA 38 TGTGTCTCTA GTGACTGTCA CTCG24 42 base pairs nucleic acid single linear cDNA 39 GATCCTCAGGCTGCTGAGCT CCATGTAGGC TGTGTTTGTG GA 42 190 base pairs nucleic acid bothlinear cDNA 40 CACATCCACA AGCTTAAACA CAGCCGAGCT CAGCAGCCTG AGGTCTGAGGACACAGCCGT 60 CTATTACTGT GCAAGATCCT ACGACTTTGC CTGGTTTGCT TACTGGGGCCAAGGGACTC 120 GGTCACAGTC TCCTCAGGTG AGTCCTTACA ACCTCTCTCT TCTATTCAGTCGACATAGA 180 ACGTGGATCC 190 39 base pairs nucleic acid single linearcDNA 41 AGCTTAAACA CAGCCGAGCT CAGCAGCCTG AGGTCTGAG 39 27 base pairsnucleic acid single linear cDNA 42 GACACAGCCG TCTATTACTG TGCAAGA 27 39base pairs nucleic acid single linear cDNA 43 TCCTACGACT TTGCCTGGTTTGCTTACTGG GGCCAAGGG 39 39 base pairs nucleic acid single linear cDNA 44ACTCTGGTCA CAGTCTCCTC AGGTGAGTCC TTACAACCT 39 31 base pairs nucleic acidsingle linear cDNA 45 CTCTCTTCTA TTCAGTCGAC ATAGATACGT G 31 17 basepairs nucleic acid single linear cDNA 46 GAGCTCGGCT GTGTTTA 17 33 basepairs nucleic acid single linear cDNA 47 ATAGACGGCT GTGTCCTCAGACCTCAGGCT GCT 33 39 base pairs nucleic acid single linear cDNA 48GTAAGCAAAC CAGGCAAAGT CGTAGGATCT TGCACAGTA 39 36 base pairs nucleic acidsingle linear cDNA 49 ACCTGAGGAG ACTGTGACCA GAGTCCCTTG GCCCCA 36 29 basepairs nucleic acid single linear cDNA 50 TGAATAGAAG AGAGAGGTTG TAAGGACTC29 21 base pairs nucleic acid single linear cDNA 51 GATCCACGTATCTATGTCGA C 21 24 base pairs nucleic acid single linear cDNA 52GATAGACAGA TGGGGGTGTC GTTT 24 24 base pairs nucleic acid single linearcDNA 53 AGATGGATAC AGTTGGTGCA GCAT 24 18 base pairs nucleic acid singlelinear cDNA 54 TGTCATTGGT ACCCATAT 18 21 base pairs nucleic acid singlelinear cDNA 55 AAATCTATGT CGACTGAATA G 21 75 base pairs nucleic acidsingle linear cDNA 56 CTGCTGGTAC CAGGCCAAGT AGATCTTTTG ATTGCTACTATATAAAAGGC TCTGACTGGA 60 CTTACAGGTG ATGGT 75 45 base pairs nucleic acidsingle linear cDNA 57 GCTTGGCACA CCAGATTCCC TAGTGGATGC CCAGTAGATC AGCAG45 51 base pairs nucleic acid single linear cDNA 58 CCCTTGGCCGAACGTCCGAG GATATCTATA ATATTGCTGG CAGTAGTAGG T 51 354 base pairs nucleicacid both linear cDNA 59 CAGGTGCAGC TGGTGCAGTC TGGGGCAGAG GTGAAAAAGCCTGGGGCCTC AGTGAAGGTG 60 TCCTGCAAGG CTTCTGGCTA CACCTTCAGT GCCTACTGGATAGAGTGGGT GCGCCAGGC 120 CCAGGAAAGG GCCTCGAGTG GGTCGGAGAG ATTTTACCTGGAAGTAATAA TTCTAGATA 180 AATGAGAAGT TCAAGGGCCG AGTGACAGTC ACTAGAGACACATCCACAAA CACAGCCTA 240 ATGGAGCTCA GCAGCCTGAG GTCTGAGGAC ACAGCCGTCTATTACTGTGC AAGATCCTA 300 GACTTTGCCT GGTTTGCTTA CTGGGGCCAA GGGACTCTGGTCACAGTCTC CTCA 354 118 amino acids amino acid single linear protein 60Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Al 1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ala Ty 20 25 30Trp Ile Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Va 35 40 45Gly Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Ph 50 55 60Lys Gly Arg Val Thr Val Thr Arg Asp Thr Ser Thr Asn Thr Ala Ty 65 70 7580 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cy 85 9095 Ala Arg Ser Tyr Asp Phe Ala Trp Phe Ala Tyr Trp Gly Gln Gly Th 100105 110 Leu Val Thr Val Ser Ser 115 342 base pairs nucleic acid bothlinear cDNA 61 GACATCCAGA TGACCCAGAG CCCAAGCAGC CTGAGCGCCA GCGTGGGTGACAGAGTGACC 60 ATCACCTGTA AGTCCAGTCA GAGCCTTTTA TATAGTAGCA ATCAAAAGATCTACTTGGC 120 TGGTACCAGC AGAAGCCAGG TAAGGCTCCA AAGCTGCTGA TCTACTGGGCATCCACTAG 180 GAATCTGGTG TGCCAAGCAG ATTCAGCGGT AGCGGTAGCG GTACCGACTTCACCTTCAC 240 ATCAGCAGCC TCCAGCCAGA GGACATCGCC ACCTACTACT GCCAGCAATATTATAGATA 300 CCTCGGACGT TCGGCCAAGG GACCAAGGTG GAAATCAAAC GT 342 114amino acids amino acid single linear protein 62 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Val Gl 1 5 10 15 Asp Arg Val Thr Ile ThrCys Lys Ser Ser Gln Ser Leu Leu Tyr Se 20 25 30 Ser Asn Gln Lys Ile TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Ly 35 40 45 Ala Pro Lys Leu Leu IleTyr Trp Ala Ser Thr Arg Glu Ser Gly Va 50 55 60 Pro Ser Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Phe Th 65 70 75 80 Ile Ser Ser Leu GlnPro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gl 85 90 95 Tyr Tyr Arg Tyr ProArg Thr Phe Gly Gln Gly Thr Lys Val Glu Il 100 105 110 Lys Arg

1. A synthetic specific binding agent having specificity for humanpolymorphic epithelial mucin (PEM), conferred by the presence of one ormore of the amino acid sequences: i) Ala Tyr Trp Ile Glu ii) Glu Ile LeuPro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly iii) Ser Tyr AspPhe Ala Trp Phe Ala Tyr iv) Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser AsnGln Lys Ile Tyr Leu Ala v) Trp Ala Ser Thr Arg Glu Ser vi) Gln Gln TyrTyr Arg Tyr Pro Arg Thr 2 A reshaped human antibody, or a reshaped humanantibody fragment, having specificity for human polymorphic epithelialmucin (PEM) conferred by the presence of one or more of the amino acidsequences: i) Ala Tyr Trp Ile Glu ii) Glu Ile Leu Pro Gly Ser Asn AsnSer Arg Tyr Asn Glu Lys Phe Lys Gly iii) Ser Tyr Asp Phe Ala Trp Phe AlaTyr iv) Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr LeuAla v) Trp Ala Ser Thr Arg Glu Ser vi) Gln Gln Tyr Tyr Arg Tyr Pro ArgThr
 3. A reshaped human antibody or reshaped human antibody fragmentaccording to claim 2, having at least one heavy-chain variable regionincorporating the following CDRs: CDR1: Ala Tyr Trp Ile Glu CDR2: GluIle Leu Pro Gly Ser Asn Asn Ser Arg Tyr Asn Glu Lys Phe Lys Gly CDR3:Ser Tyr Asp Phe Ala Trp Phe Ala Tyr
 4. A reshaped human antibody orreshaped human antibody fragment according to claim 2, having at leastone light-chain variable region incorporating the following CDRs: CDR1:Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu AlaCDR2: Trp Ala Ser Thr Arg Glu Ser CDR3: Gln Gln Tyr Tyr Arg Tyr Pro ArgThr
 5. A reshaped human antibody or reshaped human antibody fragmentaccording to claim 2 and having at least one heavy-chain variable regionaccording to claim 3 and at least one light-chain variable regionaccording to claim
 4. 6. A reshaped human antibody or reshaped humanantibody fragment according to claim 2, incorporating at least oneheavy-chain variable region comprising the entire amino acid sequencedepicted in FIG. 12 of the accompanying drawings.
 7. A reshaped humanantibody or reshaped human antibody fragment according to claim 2,incorporating at least one light-chain variable region comprising theentire amino acid sequence depicted in FIG. 13 of the accompanyingdrawings.
 8. A synthetic specific binding agent, reshaped human antibodyor reshaped human antibody fragment, according to any one of thepreceding claims, wherein the PEM is human milk fat globule (HMFG).
 9. Asynthetic specific binding agent, reshaped human antibody or reshapedhuman antibody fragment, having specificity equivalent to that of thegamma-1, kappa anti-HMFG monoclonal antibody “HMFG1”.
 10. A stable hostcell line producing a synthetic specific binding agent, reshaped humanantibody or reshaped human antibody fragment according to any one ofclaims 1 to 9, resulting from incorporation in the cell line of aforeign gene encoding the synthetic specific binding agent, reshapedhuman antibody or reshaped human antibody fragment.
 11. A stable hostcell line according to claim 10, wherein the foreign gene includes oneor more of the nucleotide sequences: i) GCC TAC TGG ATA GAG ii) GAG ATTTTA CCT GGA AGT AAT AAT TCT AGA TAC AAT GAG AAG TTC AAG GGC iii) TCC TACGAC TTT GCC TGG TTT GCT TAC iv) AAG TCC AGT CAG AGC CTT TTA TAT AGT AGCAAT CAA AAG ATC TAC TTG GCC v) TGG GCA TCC ACT AGG GAA TCT vi) CAG CAATAT TAT AGA TAT CCT CGG ACG
 12. A stable host cell line according toclaim 10, wherein the foreign gene includes the entire nucleotidesequence depicted in FIG. 12 of the accompanying drawings.
 13. A stablehost cell line according to claim 10, wherein the foreign gene includesthe entire nucleotide sequence depicted in FIG. 13 of the accompanyingdrawings.
 14. A stable host cell line according to claim 10, wherein theforeign gene encodes: a) at least one of the amino acid sequences: i)Ala Tyr Trp Ile Glu ii) Glu Ile Leu Pro Gly Ser Asn Asn Ser Arg Tyr AsnGlu Lys Phe Lys Gly iii) Ser Tyr Asp Phe Ala Trp Phe Ala Tyr iv) Lys SerSer Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Ile Tyr Leu Ala v) Trp AlaSer Thr Arg Glu Ser vi) Gln Gln Tyr Tyr Arg Tyr Pro Arg Thr and b) aprotein framework that enables the encoded amino acid sequence whenexpressed to function as a CDR having specificity for PEM.
 15. A stablehost cell line according to claim 10, wherein the foreign gene encodesthe entire amino acid sequence depicted in FIG. 12 of the accompanyingdrawings.
 16. A stable host cell line according to claim 10, wherein theforeign gene encodes the entire amino acid sequence depicted in FIG. 13of the accompanying drawings.
 17. Plasmid pSVgpt-HuVHHMFG1-HuIgG1. 18.Plasmid pSVneo-HuVkHMFG1-HuCk.
 19. Use of plasmid according to claim 17or claim 18 in the production of a synthetic specific binding agent,reshaped human antibody or reshaped human antibody fragment.
 20. E.coliNCTC
 12411. 21. E.coli NCTC
 12412. 22. A DNA sequence encoding areshaped human antibody heavy-chain variable region having specificityfor HMFG, as contained in E.coli NCTC
 12411. 23. A DNA sequence encodinga reshaped human antibody light-chain variable region having specificityfor HMFG, as contained in E.coli NCTC
 12412. 24. A reshaped humanantibody heavy-chain variable region having specificity for HMFG,producible by means of the expression vector contained in E.coli NCTC12411.
 25. A reshaped human antibody light-chain variable region havingspecificity for HMFG, producible by means of the expression vectorcontained in E.coli NCTC
 12412. 26. A reshaped human antibody orreshaped human antibody fragment, comprising at least one variableregion according to claim 24 or claim
 25. 27. A synthetic specificbinding agent, reshaped human antibody or reshaped human antibodyfragment, according to any one of claims 1 to 9 or claim 26, linked toor incorporating an agent capable of retarding or terminating the growthof cancerous cells, or linked to an agent capable of being detectedwhile inside the human body.
 28. An injectable composition comprising asynthetic specific binding agent, reshaped human antibody or reshapedhuman antibody fragment, according to claim 27, in a pharmaceuticallyacceptable carrier.
 29. Use of a synthetic specific binding agent,reshaped human antibody or reshaped human antibody fragment, accordingto any one of claims 1 to 9 or claim 26, for the manufacture of amedicament for therapeutic application in the relief of cancer inhumans, or for the manufacture of a diagnostic composition for in-vivodiagnostic application in humans.
 30. Use of a synthetic binding agent,reshaped human antibody or reshaped human antibody fragment, accordingto claim 27, in a method of human cancer therapy or imaging.